Isolated species of steinernematid nematode and methods of white grub control therewith

ABSTRACT

An isolated Steinernematid nematode of the species  Steinernema scarabaei  (ATCC accession No. PTA-6988 is provided which is entomopathogenic to the larvae of scarab beetles, e.g., Japanese beetle ( Popillia japonica ), oriental beetles ( Exomala  (= Anomala )  orientalis ), European chafer ( Rhizotrogus majalis ), Asiatic garden beetle ( Maladera castanea ), masked chafers ( Cyclocephala  spp.), and May/June beetles ( Phyllophaga  spp.). A biopesticide composition is provided which comprises an insecticidally effective amount of  Steinernema scarabaei.  Biopesticide compositions are described which, in addition to  Steinernema scarabaei,  further comprise at least one neonicotinoid insecticide, e.g., imidacloprid. Methods are provided for controlling the larvae of at least one species of scarab beetle comprising applying a biopesticide composition to the locus of the larvae wherein the composition comprises an insecticidally effective amount of an isolated entomopathogenic nematode of the species  Steinernema scarabaei (ATCC accession No. PTA-6988).

Priority is claimed under Title 35 of the United States Code § 119(e)from U.S. provisional application Ser. No. 60/315,335, which was filedAug. 29, 2001.

FIELD OF THE INVENTION

This invention relates to a novel entomopathogenic nematode of the genusSteinernema, which is effective as a biopesticide for the control ofinsects, particularly the larvae of scarab beetles such as Japanesebeetle (Popillia japonica), oriental beetles (Exomala (=Anomala)orientalis), European chafer (Rhizotrogus majalis), Asiatic gardenbeetle (Maladera castanea), masked chafers (Cyclocephala spp.), andMay/June beetles (Phyllophaga spp.). Biopesticide compositions areprovided which comprise an insecticidally effective amount of anisolated nematode of the species Steinernema scarabaei (ATCC accessionNo. PTA-6988). Methods are provided for controlling the larvae of scarabbeetles.

BACKGROUND OF THE INVENTION

White grubs, the root-feeding larvae of scarab beetles (Coleoptera:Scarabaeidae), are important pests of turf and pasture grasses,ornamental plants, and numerous crops around the world. At least 10species cause significant damage to turfgrasses in North America. Infact, a complex of primarily introduced white grub species are the majorturfgrass insect pests. Among these, the Japanese beetle, Popilliajaponica, has until recently been regarded as the key species, but otherwhite grub species are becoming more important. These other speciesinclude the oriental beetle, Exomala orientalis, the European chafer,Rhizotrogus majalis, and the Asiatic garden beetle, Maladera castanea.Surveys have indicated that the oriental beetle has become the mostimportant white grub species. The subterranean habit of the larvae ofscarab beetles makes them some of the most difficult to control insectpests.

Chemical insecticides are the primary means of controlling white grubs.Organophosphate and carbamate insecticides are used for curative controlof white grubs, however, toxicological and environmental problemsrelated to their application have already or will soon lead to the lossof registrations of many compounds or uses of these compounds. Theimplementation of the Food Quality Protection Act of 1996 in the UnitedStates, for example, is responsible for these increasing restrictions onthe use of organophosphates (and potentially also carbamates). While newtypes of insecticides with better toxicological and environmentalcharacteristics (e.g., neonicotinoids, insect growth regulators) arebecoming increasingly available for white grub control, these compoundsgenerally have to be applied preventively because their efficacydeclines with advancing white grub development. Because white gruboutbreaks are generally difficult to predict, this preventive approachoften results in the treatment of large areas that may only need partialor no treatment. This not only increases the cost of white grubmanagement, but also may have unintended environmental consequences suchas long-term suppression of beneficial insects.

Entomopathogenic nematodes (Heterorhabditidae and Steinernematidae)offer an environmentally safe ‘biopesticide’ alternative to chemicalinsecticides for the control of white grubs. These nematodes possessmost of the characteristics of an ideal biological control agent forinsects. Koppenhöfer, In: Lacey and Kaya (eds.), Field Manual ofTechniques in Invertebrate Pathology, Kluwer Academic Publishers, pp.283-301 (2000). Nematodes have been studied quite extensively as whitegrub control agents. However, overall the level of white grub controlhas been often inconsistent and unsatisfactory. Klein, In: Nematodes andthe biological control of insect pests, CSIRO, East Melbourne, pp. 49-58(1993). A reason for this inconsistency is the difference in nematodesusceptibility among different white grub species. Thus, important whitegrub pests such as the European chafer or the oriental beetle, forexample, are very resistant to infection by some of the previously usednematode species.

SUMMARY OF THE INVENTION

The present invention is directed to an isolated entomopathogenicnematode of the species Steinernema scarabaei (ATCC accession No.PTA-6988). which is an effective biopesticide agent for the control ofinsects, particularly the larvae of scarab beetles such as the Japanesebeetle (Popillia japonica), oriental beetles (Exomala (=Anomala)orientalis), European chafer (Rhizotrogus majalis), Asiatic gardenbeetle (Maladera castanea), masked chafers (Cyclocephala spp.), andMay/June beetles (Phyllophaga spp.).

The invention is further directed to an isolated and substantiallyhomogenous population of a nematode of the species Steinernemascarabaei.

A biopesticide composition is provided which comprises an insecticidallyeffective amount of an isolated nematode of the species Steinernemascarabaei.

A biopesticide composition is further provided which comprises aninsecticidally effective amount of an isolated nematode of the speciesSteinernema scarabaei—and—at least one neonicotinoid insecticide, e.g.,imidacloprid.

An object of the invention is to provide a method for controlling thelarvae of scarab beetles (white grubs) comprising applying abiopesticide composition to the locus of the larvae wherein thecomposition comprises an insecticidally effective amount of an isolatedentomopathogenic nematode of the species Steinernema scarabaei.

A further object of the invention is to provide a method for controllingthe larvae of scarab beetles (white grubs) comprising applying abiopesticide composition to the locus of the larvae wherein thecomposition comprises an insecticidally effective amount of an isolatedentomopathogenic nematode of the species Steinernema scarabaei—and—atleast one neonicotinoid insecticide, e.g., imidacloprid.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 displays the isolated and characterized Steinernema species ofthe present invention; wherein, view A shows an entire body of firstgeneration male; view B shows an anterior end (lateral view) of firstgeneration male; view C shows spicule and gubernaculum of firstgeneration male; view D shows a tail (lateral view) of first generationmale; view E shows an entire body of first generation female; view Fshows a vulva (lateral view) of a first generation female; view G showsa tail (lateral view) of a first generation female; view H shows ananterior end (lateral view) of third-stage infective juvenile; and, viewI shows a tail (lateral view) of a third-stage infective juvenile.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs. All publications and patentsreferred to herein are incorporated by reference.

The term “entomopathogenic nematode” is used in the art to refer to thenematode's ability to quickly kill hosts facilitated by theirmutualistic (symbiotic) association with bacteria. The stage thatsurvives outside of a host is the non-feeding, non-developing thirdstage infective juvenile (IJ) or dauer juvenile. The infective juvenilescarry cells of their bacterial symbiont in their intestines. Afterlocating a suitable host, the infective juveniles invade it throughnatural openings (mouth, spiracles, anus) or thin areas of the host'scuticle and penetrate into the host hemocoel. The infective juvenilesrelease their symbiotic bacteria that propagate, kill the host bysepticemia, and metabolize its tissues. The nematodes start developingand feed on the bacteria and metabolized host tissues, and go through1-3 generations, until a new generation of infective juveniles emergesfrom the depleted host cadaver.

An “isolated” Steinernematid nematode species, for example, as usedherein refers the characterized species of the present inventiondescribed herein, Steinernema scarabaei, American Type CultureCollection (ATCC), 10801 University Blvd., Manassas, Va. 20110 - USAacession No. PTA-6988, deposited Sep. 20, 2005, which is artificiallyseparated and particularly cultivated, propagated and/or stored apartfrom its natural soil environment. Although the species name is not yetofficial, the isolated and characterized Steinernema species of thepresent invention described herein will be referred to as Steinernemascarabaei (ATCC accession No. PTA-6988.

An “insecticidally effective amount” is defined herein as that quantitythat results in a significant mortality rate of the target insects understandard laboratory conditions, for example, as demonstrated in theExamples and Tables presented herein—or—a 5 ft.×5 ft. field test locus,for example, when compared to a control or untreated group. Aninsecticidally effective amount may refer to an approximate number ofSteinernema scarabaei infective juveniles (IJ) per hectare (ha), forexample, that may be necessary to control the larvae of at least onespecies of scarab beetle, e.g., Japanese beetle (Popillia japonica),oriental beetles (Exomala (=Anomala) orientalis), European chafer(Rhizotrogus majalis), Asiatic garden beetle (Maladera castanea), maskedchafers (Cyclocephala spp.), and May/June beetles (Phyllophaga spp.).

An isolated and substantially homogenous population of a nematode of thespecies Steinernema scarabaei refers to a population which isartificially separated and preferably stored apart from its natural soilenvironment and which sample is particularly suitable for cultivation orseeding propagation or production of the species.

Steinernema scarabaei of the current invention is demonstrated to showunusually high pathogenicity toward particular beetle grubs (furtherdescribed infra). Moreover the size, morphological features, andbehavior of the infective juvenile stage of the species is distinct(further described infra) from entomopathogenic nematode speciescurrently known. The Steinernema scarabaei of the current invention doesnot cross-hybridize or inter-breed with any other known species.

Commercial Utility

This invention relates to a novel entomopathogenic nematode of the genusSteinernema, which is effective as a biopesticide for the control ofinsects, and particularly the larvae of scarab beetles (white grubs),including but not limited to, the Japanese beetle (Popillia japonica),oriental beetles (Exomala (=Anomala) orientalis), European chafer(Rhizotrogus majalis), Asiatic garden beetle (Maladera castanea), maskedchafers (Cyclocephala spp.), and May/June beetles (Phyllophaga spp.). Asignificantly valuable aspect of the current invention is thepathogenicity of Steinernema scarabaei to white grubs and demonstratedefficacy, particularly in controlling the larvae of the Japanese beetle.This provides for commercial embodiments of compositions and methods ofuse for controlling the larvae of these and other scarab beetles. Thenematode may be applied for the control of economically importantinsects on turf and pasture grasses, ornamental plants, or any crop thatmay be damaged by these pests. When applied to the locus of the targetinsects described herein, for example, Steinernema scarabaei of theinvention will provide very good to complete suppression or control ofthe target insect population.

The Steinernema scarabaei of the present invention is a pathogen highlyadapted to the larvae of scarab beetles as an integral element of itsown reproductive cycle. In fact, as demonstrated in the Examples andTables presented herein, the pathogenicity of the Steinernema species ofthis invention herein is significantly greater, for example, against thelarvae of Japanese beetle (Popillia japonica), oriental beetles (Exomala(=Anomala) orientalis), European chafer (Rhizotrogus majalis), Asiaticgarden beetle (Maladera castanea), masked chafers (Cyclocephala spp.),and May/June beetles (Phyllophaga spp.) than that of other presentlyknown nematodes species. The superior insecticidal properties ofSteinernema scarabaei against the white grub larvae of Japanese beetles,for example, is apparent at low application rates under fieldconditions. The insecticidal efficacy of Steinernema scarabaei againstlarvae of the northern masked chafer under field conditions, forexample, is significantly greater than that of other known nematodespecies. The insecticidal efficacy of Steinernema scarabaei against thelarvae of the oriental beetle, the European chafer, and the Asiaticgarden beetle, is also several times greater than other nematodespecies.

Example II demonstrates Steinernema scarabaei infectivity compared toother nematode species/isolates. The insecticidal efficacy ofSteinernema scarabaei against the larvae of the oriental beetle, theAsiatic garden beetle, and the European chafer, for example, greatlyoutperforms any other nematode isolate. Overall, as shown in Table 1,Steinernema scarabaei is the most pathogenic nematode species/isolateunder the laboratory conditions used.

Example III further illustrates the high pathogenicity of Steinernemascarabaei. As demonstrated, only 17.9 infective juveniles (IJs) perlarva are necessary to kill 50% of oriental beetle larvae (35.2 arenecessary to kill 90%). Japanese beetle larvae are even more susceptibleand only 10.9 IJs per larva are necessary to achieve 50% mortality (22.3are necessary to achieve 90% mortality). LC50 (LC90) is a concentrationof Steinernema scarabaei that kills 50 (90) percent of a population.

Example IV provides characteristics of the infection and reproductivebiology of the species and further shows the high pathogenicity (85-98%mortality) against the larvae of the oriental beetle. All dosages ofSteinernema scarabaei caused high oriental beetle mortality (85-98%)(Table 3).

Example V demonstrates the insecticidal efficacy of Steinernemascarabaei, particularly against Japanese beetle larvae, wherein forexample at the extraordinarily low rate of 0.3125×10⁹ infectivejuveniles per ha, Steinernema scarabaei provides 90% control.Furthermore, Steinernema scarabaei causes a 2-4 times higher mortalitythan any other nematode isolate in the oriental beetle larvae.Steinernema scarabaei is also demonstrated to be effective against theAsiatic garden beetle larvae (see also, Example VII) and larvae of theEuropean chafer.

Steinernema scarabaei is shown in Example VI to provide complete control(100%) against Japanese beetle larvae within 14 days, even at thelow-end application rate of 1×10⁹ infective juvenile nematodes per ha,(Table 5) for example. Steinernema scarabaei is also demonstrated toprovide excellent control (80-93%) of oriental beetle larvae within 14days even at the lower application rate. The high infection rate ofJapanese beetle and oriental beetle larvae by Steinernema scarabaei, forexample, indicates that this newly isolated and characterized speciescan not only provide excellent short term control of scarab larvae butalso has a high potential for long-term suppression due to efficientreproduction in its hosts.

Steinernema scarabaei is also shown in Example VIII to provide control(e.g., 84%) against the northern masked chafer under field conditions.

Example IX demonstrates that the combination of Steinernema scarabaeiwith the neonicotinoid insecticide imidacloprid results in a synergisticenhancement of the mortality of the larvae of scarab beetles compared toimidacloprid alone or Steinernema scarabaei alone (Table 9). Theinteractions are synergistic in all combinations tested. Imidaclopridalone did not cause significant mortality.

Examples X, XI and XII are directed toward the Steinernema scarabaeitaxonomic description of Females, Males, and Third stage infectivejuveniles, respectively.

Morphological Features

Morphological data indicate that Steinernema scarabaei belongs to thegroup of species commonly know as the S. glaseri-group. Steinernemascarabaei can be separated from other Steinernema spp. by severalcharacteristics. The average IJ body length of the Steinernema scarabaeidescribed herein is about 918 μm, and ranges from about 890 μm to about959 μm. The average maximum body width of each Steinernema scarabaeisingle specimen is about 31 μm (the range of diversity is from about 25μm to about 37 μm).

The new species can be distinguished from most of the taxa that belongto the S. glaseri-group, for example, by the average body length of theIJs, which is shorter than that of S. arenarium, S. glaseri, S.longicaudum, S. puertoricense and S. cubanum. The nerve ring of the IJof the Steinernema scarabaei described herein is more posteriorlylocated (about 120 μm from the anterior end of the nematode) than thatof S. feltiae (about 99 μm from the anterior end of the nematode) and S.kraussei (about 107 μn from the anterior end of the nematode).

Females of Steinernema scarabaei can be distinguished from S. arenarium,S. glaseri, S. longicaudum, S. puertoricense and S. cubanum, forexample, by the morphology of the vulval lips (slightly protruding) andtail (conoid in first generation female, blunt-conoid in secondgeneration females). Additionally, the presence of a tail papilla(located at the base of the mucro) in the females of Steinernemascarabaei is a feature that is unique to this species and has not beenreported in any previously described Steinernema spp.

The IJs of the new species resemble most closely S. karii. Somekey-diagnostic morphological characters of this stage (i.e. total bodylength, tail length, D % and E %) are very similar in these two species.However Steinernema scarabaei can be distinguished from S. karii, forexample, by the morphology of the male spicules and gubemaculum and thenumber and arrangement of the genital papillae. The size of the spiculesof Steinernema scarabaei is much shorter (having an average length ofabout 75 μm (the range of diversity is from about 67 μm to about 83 μm))than those of S. karii (average: 83 μm), but longer than that of S.cubanum (average: 58 μm) and S. kushidai (average: 63 μm). The size ofthe gubernaculum of the new species is shorter (having an average lengthof about 43.5 μm (the range of diversity is from about 36 μm to about 50μm) than that of S. arenarium (average: 53 μm) and S. glaseri (average:55 μm), but longer than that of S. cubanum (average: 39 μm). Thearrangement of Steinernema scarabaei genital papillae is different fromthat of S. glaseri, S. arenarium, S. puertoricense, S. cubanum and S.kushidai. Male Steinernema scarabaei have from about 23 to about 25genital papillae (usually 11-12 pairs and usually one single) arrangedusually as follows: 6 precloacal subventral pairs, one precloacallateral pair, one single ventral precloacal papilla (located betweenprecloacal pairs 5 and 6), one adcloacal (or postcloacal in somespecimen) pair, one postcloacal subventral pair, postcloacal subdorsalpair, and one postcloacal terminal pair near the tail tip. An additionalpair of papillae may be present at the base of the cloacal cone inaround 40% of first generation males.

Nucleic acid sequence data derived from Large Subunit of ribosomal DNA(LSU rDNA), for example, from the Steinernema scarabaei describedherein, when compared to a library of over 20 species, moreoverindicates that the Steinernema scarabaei of the invention is a distinctspecies. Phylogenetic parsimony analysis of these sequences indicatesSteinernema scarabaei is more closely related, for example, to S. kariias well as a new Steinernema species from California. However, whencompared to these two species, Steinernema scarabaei presents 19autapomporphies (unique characters). Additionally and when compared tothe “feltiae/kraussei/oregonense” group, this new species differs inmore than 30 autapomorphies. Finally, in cross-hybridization testsSteinernema scarabaei does not interbreed with any of themorphologically similar species. Reproductive compatibility of the newspecies was tested using the following Steinernema spp: Steinernemaglaseri (Steiner), S. puertoricense Román and Figueroa, S. longicaudumShen and Wang (USA isolate), S. karii Waturu, Hunt and Reid, S. kushidaiMamiya, and S. arenarium (Artyukhovsky).

The infective juvenile Steinernema scarabaei described herein can bedistinguished from other Steinernema spp. by morphological features,which include but are not limited to, for example, the position of theexcretory pore, which is about 77 μm from the anterior end of thenematode (the range of diversity is from about 72 μm to about 82 μm fromthe anterior end of the nematode). This feature, the excretory pore, ismore posteriorly located in the Steinernema scarabaei of the presentinvention than that of S. oregonense (about 66 μm from the anterior endof the nematode), S. feltiae (about 62 μm from the anterior end of thenematode) and S. kraussei (about 63 μm from the anterior end of thenematode). First-generation males of Steinernema scarabaei of theinvention can also be distinguished from S. kraussei, for example, byhaving larger spicules. First-generation male Steinernema scarabaeidescribed herein display spicules having an average length of about 75μm (the range of diversity is from about 67 μm to about 83 μm); whereas,first-generation male S. kraussei display spicules having an averagelength of about 49 μm. First-generation males of Steinernema scarabaeiof the invention can also be distinguished from S. kraussei by havinglarger gubernaculum. First-generation male Steinernema scarabaeidescribed herein display gubernaculum having an average length of about43.5 μm (the range of diversity is from about 36 μm to about 50 μm);whereas, first-generation male S. krassei display gubernaculum having anaverage length of about 33 μm. The Steinernema scarabaei describedherein IJ D %-value (distance from anterior end to excretory poredivided by esophagus length×100) of is about 60 (the range of diversityis from about 50 to about 110). Males of the new species can bedistinguished from S. kraussei by the arrangement of the male genitalpapillae, and by the absence of a mucro in the tail of the secondgeneration males. IJs of Steinernema scarabaei can be separated from S.krassei by the value of D % (average: 60 vs. 47 in S. kraussei) and E %(average: 100 vs. 80 in S. kraussei). First generation females of thenovel Steinernema scarabaei can be distinguished from all otherSteinernema spp. by the presence of of a tail papilla (located at thebase of the mucro) in first generation females that is unique to thisspecies and has not been reported from any other described Steinernemaspp.

It is noted herein however that a shift in the average as well as theranges recited herein may be accomplished by rearing the nematodes underother than normal conditions (e.g. a bad host or overcrowding).

Production

The Steinernema scarabaei, of the present invention may be isolated fromits natural environment in larvae of, for example, Japanese beetles(Popillia japonica) or oriental beetle (Exomala (=Anomala) orientalis)from the soil of turfgrass, for example. The nematode has been isolated,for example, in turfgrass areas in the northeastern United Sates.Particularly, the species of the invention can be isolated fromturfgrass soil in the state of New Jersey, for example, from larvae ofthe Japanese beetle and the oriental beetle, and from soil samples usingJapanese beetle and oriental beetle larvae as baits. The nematode may beisolated from the field by collecting infected-looking white grubs fromthe soil under turfgrass areas and placing them on emergence traps(known in the art as White traps) to collect any emerging progenynematodes from the grub cadavers. White, Science, 665:302-303, (1929).Soil samples may also be taken from the field, for example, and baitedby adding grubs as baits for the nematodes. Then grubs that becomeinfected in the soil cups may be placed onto emergence traps. SeeExample I.

Production of the nematode may be accomplished using in vivo or in vitrotechniques known in the art. As described in the Examples herein,Steinernema scarabaei may be initially recovered from infected scarablarvae recovered from the field or from soil samples using scarab larvaeas baits. Following isolation from the environment, the nematodes maythen be reared in vivo in susceptible host insects such Japanese ororiental beetle larvae (also in late instar larvae of the greater waxmoth, Galleria mellonella) as illustrated in the Examples. In accordancewith preferred methods of commercial production, for example, thenematodes may also be produced on a large scale using in vitro rearingtechniques. See, e.g., Shapiro-Ilan, D. I., et al., ProductionTechnology for Entomopathogenic Nematodes and their Bacterial Symbionts,J. Ind. Microbiol. Biotechnol., 28(3):137-46 (2002); Ehlers, R. U., MassProduction of Entomopathogenic Nematodes for Plant Protection, Appl.Microbiol. Biotechnol., 56(5-6):623-33 (2001); Friedman, et al., MassProduction in Liquid Culture of Insect-killing Nematodes, U.S. Pat. No.5,023,183, issued Jun. 11, 1991. In accordance with either in vivo or invitro techniques, the nematodes may be subsequently isolated andcollected in pure or substantially pure form. A variety of formulationsare available moreover to facilitate nematode storage. Shapiro-Ilan D I,et al., Production Technology for Entomopathogenic Nematodes and theirBacterial Symbionts, J. Ind. Microbiol. Biotechnol., 28(3):137-46(2002).

Biopesticide Compositions

Suitable formulations for commercial insecticidal biopesticidecompositions are prepared from Steinernema scarabaei nematodes isolatedfrom the environment, particularly in vitro cultivated populations ofthe nematodes, preferably substantially pure Steinernema scarabaeinematodes. Because of the moisture requirements of these nematodes forcontinued viability and infectivity, the nematodes are advantageouslyapplied in combination with water and/or another suitable inert carrieror vehicle as known in the art, which carrier is optionallysubstantially biologically pure. The term “substantially biologicallypure inert carrier” is defined herein as an inert carrier havingsignificantly fewer naturally occurring microorganisms relative to theenvironment. A preferred biopesticide composition comprises aninsecticidally effective amount of an isolated entomopathogenic nematodeof the species Steinernema scarabaei and a carrier. The carrier may bewater. Formulations may be produced that are stable for storage and,depending upon the composition, nematode viability can be maintained,for example, one year or more with refrigeration. As a practical matter,to facilitate handling and transport of the Steinernema scarabaeibiopesticide compositions, and to prevent desiccation, the formulationsof the nematode should be enclosed within a container such as a drum,jug, flask, or plastic bag as in known in the art.

A well-known variety of formulations are available to facilitatenematode application. See, e.g., Shapiro-Ilan, D. I., et al., ProductionTechnology for Entomopathogenic Nematodes and their Bacterial Symbionts,J. Ind. Microbiol. Biotechnol., 28(3): 137-46 (2002). Of particularinterest are formulations employing water as a carrier, with apopulation of the nematodes suspended therein. In an alternativeembodiment the carrier may be, or the biopesticide composition mayfurther comprise, a solid phase carrier including but not limited toencapsulating agents, upon or within which the nematodes can beimmobilized. Suitable solid phase carriers of this type include but arenot limited to hydrogel agents such as alginate gels, wheat-glutenmatrices, starch matrices, wheat-bran bait pellets, clay particles,polyacrylamide gels, or synthetic polymers as are known in the art,activated charcoal, peat, polyurethane sponge, vermiculite, and/or waterdispersible granules (WDG). WDG formulation may be used, for example, inwhich the nematodes enter a partially anhydrobic state allowing for themto survive for relatively long periods of time. See, e.g., Georgis, etal., Formulation of Entomopathogenic Nematodes, In: Hall, F. R, et al.,Eds., Biorational Pest Control Agents: Formulation and Delivery,American Chemical Society, Washington D.C., 197-205 (1995). Formulationsof alginate gels containing the nematodes provide the added benefit ofenhanced viability after storage, while allowing subsequent conversionto an aqueous liquid by dissolution of the alginate with sodium citrate.When the carrier is other than water, sufficient moisture should beprovided to ensure viability and infectivity of the nematodes. Besidesthe active agent itself, other additives and adjuncts may be formulatedinto the compositions of the invention. Examples of these includenutrients, humectants, feeding stimulants (phagostimulants), UVprotectants, inert fillers, and dispersants. Humectant materials includebut are not limited to glycerol, sugars such as sucrose, invertemulsions, and cellulose ethers. See, e.g., Nelson, et al., U.S. Pat.No. 4,701,326; Georgis, R., Formulation and Application Technology, In:Gaugler, et al., Eds., Entomopathogenic Nematodes in Biological Control,CRC Press, Boca Raton, Fla., 173-194 (1990); Raulston, et al., U.S. Pat.No. 6,184,434.

Methods of Use

An insecticidally effective amount of the Steinernema scarabaei isapplied to the locus of, or in the vicinity of, insects to becontrolled. An insecticidally effective amount for example may refer toan approximate number of Steinernema scarabaei infective juveniles (IJ)per hectare (ha) that may be necessary to control the larvae of at leastone species of scarab beetle, e.g., Japanese beetle (Popillia japonica),oriental beetles (Exomala (=Anomala) orientalis), European chafer(Rhizotrogus majalis), Asiatic garden beetle (Maladera castanea), maskedchafers (Cyclocephala spp.), and May/June beetles (Phyllophaga spp.).The actual effective amount may be readily determined by thepractitioner skilled in the art, and may vary with the species of pest,stage of larval development, the type of vehicle or carrier, the periodof treatment, environmental conditions (especially moisture), and otherrelated factors. Without being limited thereto, in accordance withembodiments of the invention, the Steinernema scarabaei nematodes aregenerally applied at a concentration of—from about 0.25×10⁹ infectivejuveniles per hectare in the field—to about 5×10⁹ infective juvenilesper hectare in the field. However, less (or more) can be used dependingupon the compositions, including the combination of Steinernemascarabaei with imidacloprid, for example; and formulations employed, aswell as conditions, locus, identity, and population of the targetinsects.

In accordance with an embodiment for use in areas employing irrigation,the nematodes may be admixed with irrigation water prior to or at thetime of irrigation, effectively distributing the nematodes across thefield.

A preferred method of the invention is that of controlling the larvae ofat least one species of scarab beetle comprising applying a biopesticidecomposition of the invention to the locus of the larvae to be controlledand wherein the composition comprises an insecticidally effective amountof an isolated entomopathogenic nematode of the species Steinernemascarabaei.

The field (locus) rate of application of the newly isolated andcharacterized species, Steinernema scarabaei, for controlling the larvaeof scarab beetles, particularly the Japanese beetle, oriental beetle,European chafer, Asiatic garden beetle and/or masked chafer species, forexample, ranges generally from about 0.25×10⁹ infective juveniles (IJ)per hectare (ha) to about 5×10⁹ IJ per ha. A preferred range ofSteinernema scarabaei application is from about 1×10⁹ IJ per ha to about2.5×10⁹ IJ per ha. This preferred range is also a preferred range ofrate of Steinernema scarabaei application to control the larvae ofscarab beetles, particularly the Japanese beetle, oriental beetle,European chafer, Asiatic garden beetle and masked chafer species, forexample, in turfgrass. However, lower rates of Steinernema scarabaeiapplication may be used, for example, from about 0.25×10⁹ IJ per ha toabout 1×10⁹ IJ per ha, as well as higher rates, for example, from about2.5×10⁹ IJ per ha to about 5×10⁹ IJ per ha, may be successfully employedfor controlling the larvae of scarab beetles as described herein. Theserates of application are intended to be within the scope of the claimsappended hereto.

Example V demonstrates the insecticidal efficacy of Steinernemascarabaei, particularly against Japanese beetle larvae, wherein forexample at the extraordinarily low rate of 0.3125×10⁹ infectivejuveniles per ha, Steinernema scarabaei provides 90% control. Which ratewill provide acceptable control will depend on a number factorsincluding white grub species to be controlled, crop system andmanagement practices, environmental conditions, degree of controlneeded, and speed of control required.

In order to maximize the efficacy of the insecticidal activity ofSteinernema scarabaei, and the biopesticide compositions/formulationsdescribed herein, because Steinernema scarabaei actively seek out andthen penetrate and parasitize the target insects, the application of thenematodes and compositions to the soil should be properly timed to thepresence of scarab beetle larvae, for example, in the soil when they, asa group population, are most susceptible.

Combinations

Combining nematodes with synergists may be a strategy that could permitcost-effective use of nematodes, even under stringent situations. Anefficient combination with wide applicability is that of Steinernemascarabaei and the neonicotinoid insecticide imidacloprid. Koppenhöfer,et al., for example, have shown that combined applications of thescarab-adapted entomopathogenic nematodes S. glaseri or H. bacteriophoraand imidacloprid resulted in synergistic mortality of third-instar whitegrubs. See, Journal of Economic Entomology, 91:618-623 (1998);Biological Control, 19:245-251 (2000). This interaction was observedover a range of imidacloprid rates, with simultaneous or delayednematode application, and for five scarab species (P. japonica, E.orientalis, and the masked chafers, Cyclocephala hirta, C. pasadenae,and C. borealis) with different degrees of nematode susceptibility.Koppenhöfer, et al., further showed that the major factor responsiblefor this synergistic interaction is the general disruption of normalnerve function due to imidacloprid, resulting in reduced defensiveactivity of the grubs which facilitates host attachment by infectivejuvenile nematodes and subsequent infection. Entomologia Experimentaliset Applicata, 94:283-293 (2000). In fact the major factor responsiblefor synergistic interactions between imidacloprid and entomopathogenicnematodes appears to be the general disruption of normal nerve functiondue to imidacloprid resulting in drastically reduced grub activity.Grooming and evasive behavior in response to nematode attack was alsoreduced in imidacloprid-treated grubs.

Chloronicotinyl Neonicotinoid Compounds

Any neonicotinoid compound having insecticidal properties against thelarvae of scarab beetles may be used in combination with the isolatednematode species Steinernema scarabaei of the present invention inmethods of the invention to control the larvae of scarab (white grubsincluding, but not limited to the larvae of Japanese beetles, Europeanchafers, Asiatic garden beetles, oriental beetles, May/June beetles, andmasked chafers) beetles and to provide compositions of the presentinvention. A preferred biopesticide composition of the present inventioncomprises an insecticidally effective amount of an isolatedentomopathogenic nematode of the species Steinernema scarabaei, at leastone neonicotinoid insecticide, and a carrier. However, although intendedto be within the scope of the claims appended hereto, due to thesynergistic activity of Steinernema scarabaei in combination withimidacloprid, for example, the amount of neonicotinoid insecticide, inand of itself, employed within the biopesticide compositions describedherein need not necessarily be of an “insecticidally effective amount”.

Example neonicotinoid compounds for use in the present invention includebut are not limited to, for example, imidacloprid[1-(6-chloro-3-pyridylmethyl)-N-nitroimidazolidin-2-ylideneamine, CAS138261-41-3], thiamethoxam, and acetamiprid. See, e.g., Maienfisch P.,et al., Chemistry and biology of thiamethoxam: a second generationneonicotinoid, Pest Manag Sci., 57(10):906-13 (2001); Tomizawa M., etal., Structure and diversity of insect nicotinic acetylcholinereceptors, Pest Manag Sci., 57(10):914-22 (2001).

A method of the invention is that of controlling the larvae of at leastone species of scarab beetle comprising applying a biopesticidecomposition of the invention to the locus of the larvae to be controlledand wherein the composition comprises an insecticidally effective amountof an isolated entomopathogenic nematode of the species Steinernemascarabaei and an insecticidally effective amount of at least oneneonicotinoid insecticide, e.g., imidacloprid.

Methods of Use of Combinations

The field rate of application of compositions which comprise theneonicotinoid compound imidacloprid, in combination with the newlyisolated and characterized species, Steinernema scarabaei, forcontrolling the larvae of scarab beetles, particularly the Japanesebeetle, oriental beetle, European chafer, Asiatic garden beetle andmasked chafers, for example, ranges generally from about 25 grams (g)imidacloprid per ha to about 400 g imidacloprid per ha. See, e.g.,Koppenhöfer A. M., et al., Comparison of neonicotinoid insecticides assynergists for entomopathogenic nematodes, Biol. Contr. 24, 90-97(2002). Accordingly, compositions which comprise the isolated nematodespecies Steinernema scarabaei and imidacloprid, as active ingredients,allow for an effective field rate of application generally from about0.25×10⁹ IJ Steinernema scarabaei per hectare (ha) to about 5×10⁹ IJSteinernema scarabaei per ha and from about 25 grams (g) imidaclopridper ha to about 400 g imidacloprid per ha, respectively. Preferredcompositions which comprise the isolated nematode species Steinernemascarabaei and imidacloprid, as active ingredients, allow for aneffective field rate of application from about 0.25×10⁹ IJ Steinernemascarabaei per hectare (ha) to about 2.5×10⁹ IJ Steinernema scarabaei perha and from about 25 grams (g) imidacloprid per ha to about 200 gimidacloprid per ha, respectively. Further compositions which comprisethe isolated nematode species Steinernema scarabaei and imidacloprid, asactive ingredients, allow for an effective field rate of applicationfrom about 0.5×10⁹ IJ Steinernema scarabaei per hectare (ha) to about1.5×10⁹ IJ Steinernema scarabaei per ha and from about 50 grams (g)imidacloprid per ha to about 150 g imidacloprid per ha, respectively.

The isolated and characterized species of the present invention,Steinernema scarabaei, is deposited at the American Type CultureCollection (ATCC), accession No. PTA-6988.

EXAMPLES Example I

Nematode Extraction and Culture. A previously unknown nematode of thegenus Steinernema (suggested taxonomic name: Steinernema scarabaei)(Steinernema scarabaei n. sp. (Rhabditida: Steinernematidae), a naturalpathogen of scarab beetle larvae (Coleoptera: Scarabaeidae)) wasisolated from infected Japanese beetle and oriental beetle larvaecollected from turfgrass plots at the Rutgers Research Farm in Adelphia,N.J., during epizootics of this nematode in populations of these scarabspecies. Additional nematodes were isolated by keeping field-collectedJapanese beetle larvae and oriental beetle larvae at room temperature(22-26° C.) in 30-ml plastic cups filled with soil collected from theturfgrass plots. Infected larvae were collected from the cups 7-14 daysafter exposure to the soil samples. Infected larvae from the fieldcollections or from the cup exposure were transferred to White traps(White, Science, 665:302-303, 1929) to collect the emerging progenyinfective juveniles.

The Steinernema scarabaei nematodes were cultured in the laboratory inJapanese beetle and oriental beetle larvae. Following harvest theinfective juveniles were suspended in 50 ml of water and stored in275-ml canted neck Corning tissue culture flasks at 10° C. Nematodeswere used for experiments within 1 month of harvesting.

Larvae of Japanese beetle and oriental beetle were collected frominfested turfgrass areas in Adelphia, N.J., and stored in pasteurizedsoil individually in the wells of 24-well tissue culture plates at 10°C. Before use in experiments or for nematode rearing the larvae werekept at room temperature for 2-3 days to allow for the expression ofinfection signs if they had previously been infected with nematodes. Anyscarab larvae used for rearing and any experiments described below werein the third larval stage.

Steinernema scarabaei was also reared in the late instar larvae of thegreater wax moth, Galleria mellonella. Wax moth larvae are a standardlaboratory host for most entomopathogenic nematode species, and areeasily available from commercial fish bait producers. While thepercentage of cadavers producing progeny nematodes was somewhat lessconsistent in wax moth larvae (50-90%) than in oriental and Japanesebeetle larvae (80-90%), there was no significant difference in number ofprogeny emerging per IJ-producing cadaver. The pathogenicity againstoriental beetle 3^(rd) instars of IJs produced in wax moth larvae alsodid not differ significantly from those produced in oriental beetlelarvae.

Example II

Infectivity compared to other nematode species/isolates. Overall,Steinernema scarabaei was the most pathogenic nematode species/isolate(Table 1). The infectivity of Steinernema scarabaei was determined inlarvae of 5 scarab species: Japanese beetle, oriental beetle, Asiaticgarden beetle, European chafer, and northern masked chafer. Forcomparison, 3-5 other nematode species/isolates were included in thetest: S. glaseri (NC strain), H. bacteriophora (TF strain), H.bacteriophora (R isolate, recently isolated with wax moth larvae fromsoil in Connecticut), H. bacteriophora (HO isolate, recently isolatedfrom field-infected larvae of oriental beetle and northern masked chaferin Adelphia, N.J.), and Heterorhabditis sp. (a potentially new speciesfrom Korea). The larvae were kept individually in 30-ml plastic cupsfilled with 25 g of moist (12% w/w; −7 kPa water potential) sandy loamsoil and perennial ryegrass as food. 400 infective juveniles were addedin 0.5 ml water and the cups checked for larval mortality at 7 and 14days after treatment (DAT). The experiments were conducted at roomtemperature (22-26° C.). Each treatment had 4 replicates with 10 larvaeeach. The percentage mortality data were arcsine square root transformedand subjected to an analysis of variance by scarab species using theGeneral Linear Model (GLM) procedure software of SAS (SAS Institute,Cary, N.C., 1996). Means were separated using Tukey's test (P<0.05).

The Japanese beetle was the most nematode-susceptible scarab specieswith ≧60/≧88% mortality at 7/14 DAT in all nematode treatments. At 7DAT, Steinernema scarabaei and H. bacteriophora (HO) causedsignificantly higher mortality than any other nematode (F=87.1; df=6,21; P<0.001), but at 14 DAT, mortality had increased in all nematodespecies so that only one isolate (H. bacteriophora R) had causedsignificantly less mortality than Steinernema scarabaei (F=36.9; df=6,21; P<0.001). While the 7 DAT data indicate the superiority ofSteinernema scarabaei, clearer differences could be expected at lowernematode application rates. In the less nematode-susceptible orientalbeetle, Asiatic garden beetle, and European chafer, Steinernemascarabaei outperformed any other nematode isolate with 80-100/100%mortality at 7/14 DAT compared to ≦43/≦55% at 7/14 DAT for any othernematode isolate (oriental beetle and Asiatic garden beetle: F≧24.2;df=6, 21; P<0.001. European chafer: F≧75.9; df=4, 15; P<0.001).

Example III

Dose response of Steinernema scarabaei in Japanese beetle and orientalbeetle larvae.

The larvae were kept individually in 30-ml plastic cups filled with 25 gof moist (12% w/w; −7 kPa water potential) sandy loam soil and perennialryegrass as food. Steinernema scarabaei dosages were 0, 6, 13, 20, 25,50, 100, and 200 IJs/larvae for P. japonica and 0, 13, 20, 25, 50, 100,and 200 IJs/larva for E. orientalis. Infective juveniles were added in0.5 ml water and the cups checked for larval mortality at 7 and 14 DAT.Each treatment had 4 replicates with 10 larvae each. The experiment wasconducted at room temperature (22-26° C.). The percentage mortality datawere arcsine square root transformed and subjected to an analysis ofvariance by scarab species using the General Linear Model (GLM)procedure software of SAS (SAS Institute, Cary, N.C., 1996). Means wereseparated using Tukey's test (P<0.05).

The dosage mortality data for P. japonica and E. orientalis were alsoanalyzed using probit analysis.

E. orientalis mortality by Steinernema scarabaei increased significantlyin a dose response at 7 and 14 DAT (F≧30.4; df=5, 18; P<0.001) (Table2). At a dose of 50-200 IJs/larva, mortality was 95-100% at 14 DAT.Using the dosage range of 13 to 50 IJs, the LC50 (95% fiducial limits)at 14 DAT was 17.9 (15.6-20.2) IJs/larva. The LC90 (95% fiducial limits)at 14 DAT was 35.2 (29.6-48.0) IJs/larva. P. japonica mortality alsoincreased significantly with dosage at 7 and 14 DAT (F≧32.2; df=6, 21;P<0.001) (Table 2). At a dose of 20-200 IJs/larva, mortality was 90-100%at 14 DAT. Using the dosage range of 6 to 50 IJs, the LC50 (95% fiduciallimits) at 14 DAT was 10.9 (5.8-15.5) IJs/larva. The LC90 (95% fiduciallimits) at 14 DAT was 22.3 (15.6-67.8) IJs/larva. Based on the lack ofoverlap of LC50 fiducial limits at 7 DAT (data not shown) and 14 DAT,Steinernema scarabaei was more pathogenic to P. japonica than E.orientalis.

Example IV

Effect of nematode dosage on Steinernema scarabaei infectivity anddevelopment. All dosages of Steinernema scarabaei caused high orientalbeetle mortality (85-98%) (Table 3). E. orientalis larvae were keptindividually in 30-ml plastic cups filled with 18 g of moist (12% w/w;−7 kPa water potential) sandy loam soil and perennial ryegrass as food.The experiment was conducted at room temperature (22-26° C.). 0, 25, 38,50, 100, or 200 Steinernema scarabaei infective juveniles were added in0.4 ml water and the cups checked daily for larval mortality until 14DAT. There were 40 larvae per dosage. Larvae from one half of thereplicates that died were dissected 2 days after death to count thenumber of nematodes established in the cadavers. Larvae from the otherhalf of the replicates that died were placed individually on White trapsto determine the first day of emergence and the total number of nematodeprogeny emerging from the cadavers. The number of progeny for each grubcadaver was determined by counting four subsamples under a dissectingmicroscope. For the determination of grub mortality, groups of 10 cupswere considered a replicate. For the determination of progeny emergence,individual grubs were considered replicates; grubs that produced noprogeny were not included. The percentage mortality data were arcsinesquare root transformed before analysis. Data for percentage mortality,day of host death (in DAT), number of nematodes established per cadaver,first day of emergence of nematode progeny from cadavers, and totalnumber of nematode progeny per cadaver were subjected to an analysis ofvariance using the General Linear Model (GLM) procedure software of SAS(SAS Institute, Cary, N.C., 1996). Means were separated using Tukey'stest (P<0.05).

Mortality in the control was 7.5%. All dosages of Steinernema scarabaeicaused high oriental beetle mortality (85-98%) without significantdifferences among dosages (Table 3). The time until host death occurreddecreased with nematode dosage (F=5.4; df=4, 184; P<0.001) in a linearfashion (y=5.39−9.24x; r²=0.83). Nematode establishment increased withnematode dosage (F=63.5; df=4, 77; P<0.001) in a linear fashion(y=−1.99+0.47x; r²=0.99) with mean establishment rates of 41-46%. Timingof first nematode progeny emergence from host cadavers was not affectedby dosage (P=0.48). Progeny numbers were lower at 100 infectivejuveniles per host than at 25 infective juveniles per host (F=2.6; df=4,78; P=0.04) but there was no relationship between progeny number anddosage (y=24.05−0.02x; r²=0.1).

Example V

The efficacy of Steinernema scarabaei against 5 scarab species (Japanesebeetle, oriental beetle, Asiatic garden beetle, European chafer,northern masked chafer) was determined under greenhouse conditions incomparison to various other nematode species/isolates. Japanese beetlelarvae were the most nematode susceptible species and even at the rateof 0.3125×10⁹ infective juveniles per ha, Steinernema scarabaei provided90% control. In the less nematode-susceptible oriental beetle larvaeSteinernema scarabaei caused a 2-4 times higher mortality than any othernematode isolate.

One-liter square pots (10×10×10 cm) filled with soil to a height of 9 cmwere seeded with perennial ryegrass and watered every 2-3 days until theend of the experiment. The grass was allowed to grow for 4 weeks beforeintroduction of 5 larvae per pot. The larvae were placed on the grass 3days before the start of an experiment. Larvae that had not entered intothe soil within 24 h were replaced. The temperature in the pots at a5-cm soil depth averaged 23.3±1.5° C. Treatments were applied in 50 mlof water. Pots were arranged in a completely randomized design.

Against Japanese beetle larvae treatments included Steinernema scarabaeiat 1.25, 0.3125, and 0.156×10⁹ IJs/ha, and H. bacteriophora TF at 1.25and 0.3125×10⁹ IJs/ha. Against oriental beetle larvae treatmentsincluded Steinernema scarabaei at 1.25, 0.625, 0.3125, and 0.156×10⁹infective juveniles per ha, and H. bacteriophora TF, H. bacteriophoraHO, H. bacteriophora R, S. glaseri NC, S. glaseri NJ43, andHeterorhabditis spec. each at 1.25×10⁹ IJs/ha. Against Asiatic gardenbeetle larvae treatments included Steinernema scarabaei at 2.5 and1.25×10⁹ infective juveniles per ha. Against European chafer larvaeSteinernema scarabaei was applied at 1.25×10⁹ IJs/ha. Against northernmasked chafer larvae treatments included Steinernema scarabaei at 2.5,1.25, 0.625, and 0.3125×10⁹ infective juveniles per ha, and H.bacteriophora TF, H. bacteriophora HO, and S. glaseri NJ43 each at1.25×10⁹ infective juveniles per ha. There were 7-20 pots per treatment.The number of surviving larvae was determined at 14 DAT and the datasubjected to analysis of variance by scarab species using the GeneralLinear Model (GLM) procedure software of SAS (SAS Institute, Cary, N.C.,1996). Means were separated using Tukey's test (P<0.05).

The results were similar to the laboratory observations (Example II)with respect to ranking of nematode efficacy and susceptibility ofscarab species (Table 4). Japanese beetle larvae were the most nematodesusceptible species and even at the rate of 0.3125×10⁹ infectivejuveniles per ha, Steinernema scarabaei provided 90% control. Only atthe lowest Steinernema scarabaei rate did control drop significantly(F=54.68; df=5, 51; P<0.001). In the less nematode-susceptible orientalbeetle larvae Steinernema scarabaei caused a 2-4 times higher mortalitythan any other nematode isolate (F=53.1; df=10, 139; P<0.001). Even at1/8 the application rate, Steinernema scarabaei still caused a highermortality than any other nematode isolate. In Asiatic garden beetle(F=121.2; df=2, 29; P<0.001) and European chafer larvae (F=529.4; df=1,14; P<0.001), Steinernema scarabaei provided very good and completecontrol, respectively.

Example VI

Field efficacy against Japanese and oriental beetle larvae. AgainstJapanese beetle larvae, Steinernema scarabaei provided complete control(100%) within 14 days even at the lower application rate (Table 5).Oriental beetle larvae were generally less susceptible to nematodeinfection, but Steinernema scarabaei provided excellent control (80-93%)even at 14 DAT at the lower application rate (F≧17.3; df=3, 8; P<0.001).The high infection rate of Japanese beetle and oriental beetle larvae bySteinernema scarabaei indicates that this species can not only provideexcellent short term control of scarab larvae but also has a highpotential for long-term suppression due to efficient reproduction in itshosts.

A field experiment was conducted at the Rutgers University Research Farmin Adelphia (Freehold, N.J.) in an area planted with perennial ryegrassand maintained using standard management procedures. The soil was asandy loam (67% sand, 19 silt, 14% clay, 2% organic matter, pH 6.7).Preapplication sampling showed that the site had no resident white grubor entomopathogenic nematode populations. Treatments were applied on May17, 2001 at 6 pm (soil temperature at 5 cm depth 19° C.; air temperature18° C.; cloudy) in 12.5 liter of water per m² (=12.5 mm) using awatering can. Because the soil was already moist before application noadditional irrigation was applied. Plots measured 60×60 cm with 1 mbuffer between plots. Each plot was surrounded by plastic edgingmaterial pushed 10 cm into the ground to restrict lateral movement oflarvae. There were 3 replicate plots per treatment arranged in arandomized complete block design. 20 Japanese and 20 oriental beetlelarvae were released into each plot. Larvae that did not dig into thesoil within 30 min were replaced.

Treatments were Steinernema scarabaei and H. bacteriophora TF each at2.5 and 10⁹ infective juveniles per ha. Controls received only water.The treatments were evaluated at 14 and 21 DAT by taking five 10 cm diamturf plugs (with a standard size golf hole cutter) to a depth of 7.5 cmand counting the number of surviving grubs and nematode-infected grubsfor each species. Percentage control was calculated for each data pointrelative to the average number of surviving larvae recovered from thecontrol plots. Percentage infection was calculated for each plot bydividing the number of larvae infected by the treatment nematode speciesby the total number of larvae, dead and alive, recovered. After arcsinesquare root transformation, the data were subjected to an analysis ofvariance by scarab species using the General Linear Model (GLM) ort-test (if only 2 means were compared) procedure software of SAS (SASInstitute, Cary, N.C., 1996). After GLM analysis means were separatedusing Tukey's test (P=0.05).

During the experimental period, soil temperature at 5 cm depth was18.9±1.0° C. and there was a total of 81 mm of rainfall and overheadirrigation. In the control plots the following number of live larvaewere recovered in 5 turf/soil cores: 5.0±1.2 and 5.0±1.2 Japanese beetlelarvae at 14 and 21 DAT, respectively, and 5.0±1.2 and 5.0±0.6 orientalbeetle larvae at 14 and 21 DAT, respectively. Against Japanese beetlelarvae, Steinernema scarabaei provided complete control (100%) within 14days even at the lower application rate (Table 5). Oriental beetlelarvae were generally less susceptible to nematode infection, butSteinernema scarabaei provided excellent control (80-93%) even at 14 DATat the lower application rate (F≧17.3; df=3, 8; P<0.001). H.bacteriophora TF, on the other hand, provided no oriental beetle controlat 14 DAT and only 53% even at the higher application rate and 21 DAT.

Example VII

Field efficacy against Asiatic garden beetle larvae.—A field experimentwas conducted at the Rutgers University Research Farm in Adelphia(Freehold, N.J.) in an area planted with perennial ryegrass andmaintained using standard management procedures. The soil was a sandyloam (67% sand, 19 silt, 14% clay, 2% organic matter, pH 6.7).Preapplication sampling showed that the site had no resident white grubor entomopathogenic nematode populations. Treatments were applied onJun. 5, 2001 at 11 am (soil temperature at 5 cm depth 20° C.; airtemperature 21° C.; sunny) in 12.5 liter of water per m² (=12.5 mm)using a watering can. Because the soil was already moist beforeapplication no additional irrigation was applied. Plots measured 30×30cm with 1 m buffer between plots. Each plot was surrounded by plasticedging material pushed 10 cm into the ground. There were 7 replicateplots per treatment arranged in a randomized complete block design. 19Asiatic garden beetle larvae were released into each plot. Larvae thatdid not dig into the soil within 30 min were replaced. Treatments wereSteinernema scarabaei at 2.5 and 10⁹ infective juveniles per ha and H.bacteriophora TF at 2.5×10⁹ infective juveniles per ha. The treatmentswere evaluated at 14 DAT by going through the soil of each plot to adepth of 10 cm and counting the number of surviving grubs. Percentagecontrol was calculated for each data point relative to the averagenumber of surviving larvae recovered from the control plots. Afterarcsine square root transformation, the data were subjected to ananalysis of variance using the General Linear Model (GLM) proceduresoftware of SAS (SAS Institute, Cary, N.C., 1996) and means wereseparated using Tukey's test (P<0.05).

During the experimental period, soil temperature at 5 cm depth was20.3±1.1° C. and there was a total of 85 mm of rainfall and overheadirrigation. In the control plots, 11.9±1.1 live Asiatic garden beetlelarvae were recovered at 14 DAT. Steinernema scarabaei provided goodcontrol at the higher application rate (Table 7). Control at the lowerSteinernema spec. rate was not significantly different from the controlat the higher rate. Control by H. bacteriophora TF, however, was notacceptable and was significantly lower (F=6.0; df=2, 18; P<0.01) than inboth Steinernema scarabaei treatments.

Example VIII

Field efficacy against northern masked chafer larvae. A field experimentwas conducted at the Rutgers University Research Farm in Adelphia(Freehold, N.J.) in an area planted with perennial ryegrass andmaintained using standard management procedures. The soil was a sandyloam (67% sand, 19 silt, 14% clay, 2% organic matter, pH 6.7).Preapplication sampling showed that the site had no resident white grubor entomopathogenic nematode populations. Treatments were applied onSep. 25, 2001 at 3 pm (soil temperature at 5 cm depth 23° C.; airtemperature 22° C.; sunny) in 12.5 liter of water per m² (=12.5 mm)using a watering can. Because the soil was already moist beforeapplication no additional irrigation was applied. 12 C. borealis larvaewere released into each microplot (0.05 m² surrounded by PVC pipesections pushed 10 cm into the ground to restrict lateral movement oflarvae). There were 8 replicate plots per treatment arranged in arandomized complete block design. Treatments were Steinernema scarabaeiat 2.5×10⁹ IJs/ha and 10⁹ IJs/ha and H. bacteriophora (TF strain) at2.5×10⁹ IJs/ha and 10⁹ IJs/ha. Larval survival was determined at 21 DATby searching through the soil in the microplots to a depth of 12.5 cm.Percentage control was calculated for each data point relative to theaverage number of surviving larvae recovered from the control plots.After arcsine square root transformation, the data were subjected to ananalysis of variance using the General Linear Model (GLM) proceduresoftware of SAS (SAS Institute, Cary, N.C., 1996) and means wereseparated using Tukey's test (P<0.05).

During the experiment, air temperatures averaged 15.3° C. and rainfalltotaled 38 mm. No additional overhead irrigation was supplied. Anaverage of 8.0±0.6 (range 6-10) C. borealis larvae were recovered fromthe control plots. Number of C. borealis larvae recovered variedsignificantly among treatments (F=12.6; df=9, 20; P<0.001) (Table 8).The highest Steinernema scarabaei rate provided significantly highercontrol (84%) than the lower Steinernema scarabaei rate (58%), which inturn provided significantly higher control than the higher (20%) andlower (6%) H. bacteriophora (TF strain) rate.

Example IX

Synergistic interaction of Steinernema scarabaei and the neonicotinoidinsecticide imidacloprid. Combination of Steinernema scarabaei withimidacloprid increased the mortality caused by Steinernema scarabaei inC. borealis larvae and E. orientalis larvae.

A greenhouse pot experiment was conducted to determine whether theapplication rates of Steinernema scarabaei against a susceptible(Oriental beetle) and a less susceptible (northern masked chafer) scarabspecies could be further decreased by combining it with theneonicotinoid insecticide imidacloprid. Imidacloprid has been shown tosynergize with various other entomopathogenic nematode species againstscarab larvae (Koppenhöfer & Kaya, Journal of Economic Entomology,91:618-623,1998; Koppenhöfer et al., Biological Control, 19:245-251,2000). One-liter square pots (10×10×10 cm) filled with soil to a heightof 9 cm were seeded with perennial ryegrass and watered every 2-3 daysuntil the end of the experiment. The grass was allowed to grow for 4weeks before introduction of 5 larvae per pot. The larvae were placed onthe grass 3 d before the start of an experiment. Larvae that had notentered into the soil within 24 h were replaced. The temperature in thepots at a 5-cm soil depth averaged 23.1±1.3° C. Treatments were appliedin 50 ml of water. Controls received water only. Pots were arranged in acompletely randomized design.

Treatments were based on previous observations with single controlagents against the selected scarab species. Treatments consisted of onerate of imidacloprid (200 g ai/ha; recommended field rate for scarabcontrol in turfgrass is 330-400 g ai/ha), Steinernema scarabaei at0.3125 infective juveniles per ha (both scarab species) or at 0.15625infective juveniles per ha (only oriental beetle) (recommended fieldrate for most nematodes against white grubs is 2.5 to 5.0×10⁹ infectivejuveniles per ha), and the combination of imidacloprid with eachSteinernema scarabaei rate. There were 10-20 replicate pots pertreatment. The pots were destructively sampled at 14 DAT and the numberof surviving larvae determined. The data were subjected to an analysisof variance by scarab species using the General Linear Model (GLM)procedure software of SAS (SAS Institute, Cary, N.C., 1996). Means wereseparated using Tukey's test (P<0.05).

Synergistic, additive, or antagonistic interactions between agents inthe combination treatments were determined using a χ² test (Finney,Probit Analysis, Cambridge University Press, London, 1964; McVay et al.,Journal of Invertebrate Pathology, 29:367-372, 1977; Koppenhöfer & Kaya,Journal of Economic Entomology, 91:618-623,1998). Grub mortality wascalculated by subtracting the number of surviving grubs from the numberof grubs released for each replicate and correcting for controlmortality (Abbott, Journal of Economic Entomology, 18:265-267, 1925).The expected additive proportional mortality M_(E) for thenematode—imidacloprid combinations was calculated byM_(E)=M_(N)+M_(I)(1−M_(N)) where M_(N) and M_(I) are the observedproportional mortalities caused by nematodes and imidacloprid alone,respectively. Results from a χ² test, χ²=(M_(NI)−M_(E))²/M_(E), whereM_(NI) is the observed mortality for the nematode-imidaclopridcombinations, were compared to the χ² table value for 1 df. If thecalculated χ² value exceeded the table value, a non-additive effectbetween the two agents was suspected. If the difference M_(NI)−M_(E) hada positive/negative value, a significant interaction was then consideredsynergistic/antagonistic.

Combination of Steinernema scarabaei with imidacloprid significantlyincreased the mortality caused by Steinernema scarabaei in C. borealislarvae (F=52.6; df=3, 52; P<0.001) and by both Steinernema scarabaeiapplication rates in E. orientalis larvae (F=102.8; df=5, 91; P<0.001)compared to the respective single agent treatments (Table 9). Theinteractions were synergistic in all combinations tested (χ²≧3.9; df=1;P<0.04). Imidacloprid alone did not cause significant mortality.

Example X

Steinernema scarabaei Taxonomoic Description—Female

Cuticle smooth under light microscopy, but with fine transverse striaevisible under SEM. Lateral field and phasmids inconspicuous. Headtruncated to slightly round, continuous with the body. Six lips unitedbut tips distinct, and with one labial papilla each located at posterior⅓ of metacorpus, just anterior to nerve-ring. Ovaries opposed, reflexedin dorsal position; oviduct well developed; glandular spermatheca anduterus in ventral position. Vagina short, with muscular walls. Vulvalocated near middle of body. First and second generation females withvulval lips slightly protruding. First generation female with conoidtail, with mucro and one caudal papilla located at the base of themucro. Second generation female with blunt-conoid tail, without mucro,without caudal papilla. First generation female without post-analswelling. Second generation female with post-anal swelling.

Example XI

Steinernema scarabaei Taxonomoic Description—Male

Cuticle, lip region, stoma and oesophageal region as in female. Bodycurved posteriorly, “J”-shaped when heat-killed. Single reflexed testis,consisting of germinal growth zone leading to seminal vesicle. Vasdeferens with inconspicuous walls. Spicules paired, symmetrical, curved,with ocre-brown coloration. Manubrium romboid. Lamina with rostrum(retinaculum) and 2 internal ribs. Velum present. Gubernaculum arcuate,about ¾ length of spicules. First generation male with conoid andmucronated tail. Second generation males with rounded tail withoutmucro. There are 23-25 genital papillae (11-12 pairs and one single)arranged as follows: 6 precloacal subventral pairs, one precloacallateral pair; one single ventral precloacal papilla (located betweenprecloacal pairs 5 and 6); one pair adcloacal (or postcloacal in somespecimens); one pair postcloacal subventral, one postcloacal subdorsalpair, one postcloacal terminal pair, near tail tip. An additional pairof papillae (40% of the specimens examined), at the base of the cloacalcone may be present in first generation males.

Example XI

Steinernema scarabaei Taxonomoic Description—Third Stage InfectiveJuvenile

Body slender, tapering regularly from base of oesophagus to anterior endand from anus to terminus. Lip region smooth; mouth closed. Cuticle withtransverse striae; lateral field distinct with 8 longitudinal ridges inmid-body region. Oesophagus long, narrow. Nerve-ring located at level ofisthmus. Excretory pore located about the middle of oesophagus. Basalbulb valvate. Cardia present. Anterior portion of intestine withdorsally displaced pouch containing symbiotic bacterium. Lumen ofintestine narrow; rectum long; anus distinct. Genital primordiumevident. Tail conoid with pointed terminus.

Tables

TABLE 1 Effect of nematode species/isolate on mortality of 5 scarabspecies larvae in 30-ml cups filled with soil and grass after exposureto 400 infective juvenile nematodes per cup for 7 and 14 days. NematodeP. japonica E. orientalis M. castanea C. borealis R. majalis 7 DATSteinernema  98 ± 2 a 96 ± 3 a  80 ± 7 a 35 ± 5 bc 100 ± 0 a scarabaeiS. glaseri NC  60 ± 7 c 35 ± 3 b  20 ± 7 bc 10 ± 7 cd  40 ± 7 b H.bacteriophora TF  85 ± 3 b 13 ± 5 cd  3 ± 3 c 30 ± 6 bc  8 ± 5 c H.bacteriophora R  75 ± 6 bc 13 ± 3 cd  5 ± 3 c 40 ± 0 ab  35 ± 6 b H.bacteriophora HO 100 ± 0 a 23 ± 2 bc  43 ± 5 b 75 ± 10 a —Heterorhabditis spec.  87 ± 2 b 15 ± 3 bcd  33 ± 3 b 43 ± 9 b — Control 3 ± 3 d  3 ± 3 d  3 ± 3 c  3 ± 3 d  3 ± 3 c 14 DAT Steinernema 100 ± 0a 98 ± 3 a 100 ± 0 a 45 ± 3 bc 100 ± 0 a scarabaei S. glaseri NC  88 ± 7ab 45 ± 3 b  23 ± 5 c 20 ± 4 c  43 ± 5 bc H. bacteriophora TF  93 ± 3 ab18 ± 5 c  5 ± 3 d 45 ± 3 bc  25 ± 6 c H. bacteriophora R  80 ± 4 b 20 ±4 c  5 ± 3 d 63 ± 8 b  55 ± 6 b H. bacteriophora HO 100 ± 0 a 30 ± 4 bc 55 ± 6 b 85 ± 5 a NA Heterorhabditis spec.  90 ± 0 ab 25 ± 3 bc  50 ± 7bc 60 ± 7 b NA Control  8 ± 3 c  3 ± 3 d  3 ± 3 d  5 ± 3 d  3 ± 3 d¹Means followed by same letter within columns are not significantlydifferent (P < 0.05, Tukey).

TABLE 2 Dose response of Steinernema scarabaei against Popillia japonicaand Exomala orientalis 3^(rd) instars in 30-ml plastic cups filled with25 g moist soil and grass 7 and 14 days after treatment (DAT). P.japonica E. orientalis No. Ijs 7 DAT 14 DAT 7 DAT 14 DAT 0  0.0 ± 0.0 0.0 ± 0.0  0.0 ± 0.0  0.0 ± 0.0 6 15.0 ± 2.9 c  22.5 ± 4.8 c — — 1340.0 ± 4.1 b  45.0 ± 2.9 b 25.0 ± 2.9 c  30.0 ± 4.1 d 20 85.0 ± 2.9 a 90.0 ± 4.1 a 50.0 ± 4.1 bc  52.5 ± 4.8 c 25 90.0 ± 4.1 a  95.0 ± 2.9 a57.5 ± 4.8 b  70.0 ± 4.1 b 50 95.0 ± 5.0 a 100.0 ± 0.0 a 97.5 ± 2.5 a100.0 ± 0.0 a 100 97.5 ± 2.5 a  97.5 ± 2.5 a 97.5 ± 2.5 a  97.5 ± 2.5 a200 97.5 ± 2.5 a  97.5 ± 2.5 a 87.5 ± 5.0 a  95.0 ± 5.0 a ¹Meansfollowed by same letter within columns are not significantly different(P < 0.05, Tukey).

TABLE 3 Effect of Steinernema scarabaei dosage on Exomala orientalislarvae mortality, time until host death, number of nematodes establishedper host cadaver, 1^(st) day of nematode progeny emergence from hostcadaver, and number of nematode progeny per cadaver. Nematode No. %Speed of kill establish- 1^(st) day of No. progeny IJs mortality¹ (DAT)ment emergence (×1000) 25 85 ± 7 a 5.5 ± 0.4 a 10.6 ± 1.0 c 19.0 ± 1.1 a28.5 ± 3.4 a 38 98 ± 2 a 5.1 ± 0.3 ab 16.1 ± 2.0 c 18.8 ± 0.7 a 20.3 ±2.3 ab 50 95 ± 5 a 4.7 ± 0.3 abc 20.4 ± 2.5 c 17.8 ± 0.7 a 24.7 ± 3.0 ab100 93 ± 5 a 4.1 ± 0.3 bc 43.6 ± 4.4 b 18.7 ± 1.0 a 15.2 ± 2.5 b 200 98± 2 a 3.7 ± 0.2 c 92.0 ± 6.9 a 17.2 ± 0.5 a 22.5 ± 2.9 ab ¹Controlmortality 7.5%. ²Means followed by same letter within columns are notsignificantly different (P < 0.05, Tukey).

TABLE 4 Effect of nematode species and application rate on percentagecontrol (± SEM) of 5 scarab species larvae in pots with grass in thegreenhouse at 14 days after treatment. Nematode Rate¹ P. japonica E.orientalis M. castanea C. borealis R. majalis Steinernema 25.0 — — 94 ±3 a 66 ± 7 a — scarabaei Steinernema 12.5 97 ± 3 a² 96 ± 2 a 80 ± 7 a 69± 5 a 100 ± 0 a scarabaei Steinernema 6.3 — 94 ± 3 a — 48 ± 7 ab —scarabaei Steinernema 3.1 90 ± 3 a 76 ± 4 ab — 38 ± 5 b — scarabaeiSteinernema 1.56 64 ± 5 b 68 ± 7 bc — — — scarabaei S. glaseri NC 12.5 —16 ± 5 ef — — — S. glaseri NJ43 12.5 — 32 ± 7 de —  6 ± 4 dc — H.bacteriophora 12.5 94 ± 4 a 24 ± 3 ef — 49 ± 4 ab — TF H. bacteriophora3.1 82 ± 5 ab — — — — TF H. bacteriophora R 12.5 — 34 ± 5 de — — — H.bacteriophora 12.5 — 47 ± 4 cd — 62 ± 8 ab — HO Heterorhabditis sp. 12.5— 24 ± 6 — — Control 0 14 ± 5c  9 ± 3 f  7 ± 3 b  2 ± 1 c  26 ± 4 b¹Rate in 10⁹ infective juvenile nematodes per ha (2.5 × 10⁹ = standardfield rate). ²Means followed by same letter within columns are notsignificantly different (P < 0.05, Tukey).

TABLE 5 Effect of nematode species and rate on percentage control (±SEM) of P. japonica and E. orientalis larvae in a turfgrass area at 14and 21 days after treatment (DAT). P. japonica E. orientalis NematodeRate¹ 14 DAT 21 DAT 14 DAT 21 DAT Steinernema 2.5 100 ± 0 a² 100 ± 0 a93 ± 7 a 93 ± 7 a scarabaei Steinernema 1.0 100 ± 0 a 100 ± 0 a 80 ± 0 a87 ± 7 ab scarabaei H. bacteriophora TF 2.5  73 ± 7 ab  93 ± 7 a  7 ± 7b 53 ± 13 bc H. bacteriophora TF 1.0  33 ± 13 b  40 ± 12 b  0 ± 12 b 40± 12 c ¹rate in 10⁹ infective juvenile nematodes per ha (2.5 × 10⁹ =standard field rate). ²Means followed by same letter within columns arenot significantly different (P < 0.05, Tukey).

TABLE 6 Percentage infection¹ of P. japonica and E. orientalis larvaeafter treatment with different rates of Steinernema scarabaei andHeterorhabditis bacteriophora TF in a turfgrass area. % P. japonicacontrol % E. orientalis control Nematode Rate² 14 DAT 21 DAT 14 DAT 21DAT Steinernema 2.5 100 ± 0 a³ —⁴ 94 ± 6 a — scarabaei Steinernema 1.0100 ± 0 a  — 79 ± 2 b — scarabaei H. bacteriophora TF 2.5  79 ± 2 b 67 ±33  0 ± 0 0 ± 0 H. bacteriophora TF 1.0  42 ± 9 c 28 ± 14  0 ± 0 0 ± 0¹Infection with treatment nematode species; no infections withnon-treatment nematode species were found. ²Rate in 10⁹ infectivejuvenile nematodes per ha (2.5 × 10⁹ = standard field rate). ³Meansfollowed by same letter within columns are not significantly different(P < 0.05, Tukey or t-test). ⁴Because of advanced stage of infectiononly few cadavers could be recovered at 21 DAT.

TABLE 7 Effect of nematode species and rate on percentage control (±SEM)of M. castanea larvae in a turfgrass area at 14 days after treatment.1^(st) trial Nematode Rate¹ % control Steinernema 2.5 71 ± 7 a²scarabaei Steinernema 1.0 60 ± 6 a scarabaei H. bacteriophora TF 2.5 33± 11 b ¹rate in 10⁹ infective juvenile nematodes per ha (2.5 × 10⁹ =standard field rate). ²Means followed by same letter within columns arenot significantly different (P < 0.05, Tukey).

TABLE 8 Effect of nematode species and rate on percentage control (±SEM)of C. borealis larvae in a turfgrass area at 21 days after treatment.1^(st) trial Nematode Rate¹ % control Steinernema 2.5 84 ± 6 a²scarabaei Steinernema 1.0 58 ± 10 b scarabaei H. bacteriophora TF 2.5 20± 6 c H. bacteriophora TF 1.0  6 ± 9 c ¹rate in 10⁹ infective juvenilenematodes per ha (2.5 × 10⁹ standard field rate). ²Means followed bysame letter within columns are not significantly different (P < 0.05,Tukey).

TABLE 9 Effect of combination of Steinernema scarabaei with imidaclopridon control of Exomala orientalis and Cyclocephala borealis larvae in agreenhouse pot experiment. Nematode Imidacloprid E. orientalis C.borealis Treatment rate¹ rate (g ai/ha) (% control) (% control)Imidacloprid 0.0 200 14 ± 4 d²  6 ± 3 c Steinernema 0.313 0 76 ± 7 bc 38± 6 b scarabaei Imidacloprid + 0.313 200 93 ± 3 a *³ 62 ±7 a **Steinernema scarabaei Steinernema 0.156 0 68 ± 7 c — scarabaei.Imidacloprid + 0.156 200 92 ± 3 ab ** — Steinernema scarabaei Control0.0 0  9 ± 4 d  2 ± 1 c ¹rate in 10⁹ infective juvenile nematodes per ha(2.5 × 10⁹ = standard field rate). ²Means followed by same letter withincolumns are not significantly different (P < 0.05, Tukey). ³Asterisksindicates significant synergistic interaction between the combinedcontrol agents (χ² test; */** indicate P < 0.05/0.01).

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described compositions and methods of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed compositions and modes for carrying out the invention whichare obvious to those skilled in the art or related fields are intendedto be within the scope of the following claims.

1. An insecticidally effective amount of a nematode which has beenisolated and propagated apart from its natural soil environment of thespecies Steinernema scarabaei, ATCC acession No. PTA-6988.
 2. Aninsecticidally effective amount of an isolated nematode according toclaim 1 which is entomopathogenic to the larvae of at least one speciesof scarab beetle.
 3. An insecticidally effective amount of an isolatednematode according to claim 2 which is entomopathogenic to the larvae ofJapanese beetles (Popillia japonica).
 4. An insecticidally effectiveamount of an isolated nematode according to claim 2 which isentomopathogenic to the larvae of at least one species of scarab beetleselected from the group consisting of (Japanese beetles (Popilliajaponica), Oriental beetles (Exomala (Anomala) Orientalis), Europeanchafers, (Rhizotrogus majalis), Asiatic garden beetles (Maladeracastanea), Masked chafers (Cyclocephala spp.), and May/June beetles(Phyllophaga spp.)).
 5. An isolated and substantially homogenouspopulation of a nematode of the species Steinernema scarabaei, ATCCaccession No. PTA-6988.
 6. An isolated and substantially homogenouspopulation of a nematode according to claim 5 which is entomopathogenicto the larvae of at least one species of scarab beetle.
 7. An isolatedand substantially homogenous population of a nematode according to claim6 which is entomopathogenic to the larvae of Japanese beetles (Popilliajaponica).
 8. An isolated and substantially homogenous population of anematode according to claim 6 which is entomopathogenic to the larvae ofat least one species of scarab beetle selected from the group consistingof (Japanese beetles (Popillia japonica), Oriental beetles (Exomala(Anomala) Orientalis), European chafers, (Rhizotrogus majalis), Asiaticgarden beetles (Maladera castanea), Masked chafers (Cyclocephala spp.),and May/June beetles (Phyllophaga spp.)).
 9. An isolated andsubstantially homogenous population of a nematode according to claim 8which is entomopathogenic to the larvae scarab beetles selected from thegroup consisting essentially of (Japanese beetles (Popillia japonica),Oriental beetles (Exomala (Anomala) Orientalis), European chafers,(Rhizotrogus majalis), Asiatic garden beetles (Maladera castanea),Masked chafers (Cyclocephala spp.), and May/June beetles (Phyllophagaspp.)).
 10. A method of controlling the larvae of at least one speciesof scarab beetle comprising applying a biopesticide composition to thelocus of the larvae to be controlled and wherein the compositioncomprises an insecticidally effective amount of an isolatedentomopathogenic nematode of the species Steinernema scarabaei,ATCCaccession No. PTA-6988.
 11. A method according to claim 10 forcontrolling the larvae of Japanese beetles (Popillia japonica).
 12. Amethod according to claim 10 for controlling the larvae at least onespecies of scarab beetle selected from the group consisting of (Japanesebeetles (Popillia japonica), Oriental beetles (Exomala (Anomala)Orientalis), European chafers, (Rhizotrogus majalis), Asiatic gardenbeetles (Maladera castanea), Masked chafers (Cyclocephala spp.), andMay/June beetles (Phyllophaga spp.)).